Detection of biomolecular interactions using polyfluorine tracers in diffusion ordered spectroscopy

Abstract

Biosensor technology has emerged as a significant contributor across various sectors, including military applications and healthcare. Nonetheless, these fields encounter numerous challenges, such as the detection of low-concentration protein complexes in biological samples and an insufficient understanding of protein interaction properties. To tackle these challenges, we have introduced an alternative method that involves calculating the diffusion coefficient using Diffusion Ordered Spectroscopy (DOSY) as a transducer. This technique facilitates the detection of interactions between molecules, such as proteins, by assessing the diffusion coefficient before and after their interaction, using a tracer molecule. A polyfluorine tracer, specificallys pentadecafluorooctanoyl, was employed to investigate the avidin–biotin interaction through DOSY, enabling the calculation of the diffusion coefficients for biotin-pentadecafluorooctanoyl before the interaction and for the avidin–biotin-pentadecafluorooctanoyl complex following the interaction. The measured diffusion coefficients were 1.937 × 10⁻⁸ m² s⁻¹ (unbound) and 1.12 × 10⁻¹¹ m² s⁻¹ (bound). As expected, the unbound species exhibited a significantly higher diffusion coefficient than the bound complex, consistent with the larger size and slower motion of the macromolecular assembly. Thus, the differences in these diffusion coefficients are attributed to changes in molecular weight and size, thereby confirming the avidin–biotin interaction. Additionally, the hydrodynamic radius of the avidin–biotin-pentadecafluorooctanoyl complex was determined from its diffusion coefficient, yielding a radius of 23 Å, which closely aligns with the radius of native avidin reported in existing literature. This demonstrates the method's capability to ascertain structural parameters. The application of polyfluorine tracers facilitated the acquisition of accurate and specific results without the need for a secondary antibody or further purification steps.